1. Field of the Invention
The invention relates to a device consisting of an optical waveguide, which at least has a diffractive element for coupling excitation radiation into the wave-guiding layer and on the wave-guiding layer of which there is located a second, tightly sealing layer made from a material which, at least at the support surface in the region of the guided excitation radiation, is transparent both to the excitation radiation and to the evanescently excited radiation at least to the penetration depth of the evanescent field, and which has, at least in a partial region of the guided excitation radiation, a cavity for an analysis sample, the depth of the cavity corresponding at least to the penetration depth of the evanescent field, and wherein the diffractive element is fully covered by the material of the second layer in the coupling-in region of the excitation radiation. The invention relates also to a method that uses the device for detecting, in an analyte sample, molecules that are capable of being evanescently excited to luminescence.
2. Description of the Related Art
Planar waveguides for generating and detecting evanescently excited radiation have recently been developed especially in the area of biochemical analytical science. In the evanescent field, on contact with an analyte sample, luminescence, for example fluorescence, is generated, the measurement of which allows the qualitative and quantitative determination of substances even when they are present in very low concentrations. The evanescently excited radiation emitted isotropically into space is determined by means of suitable measuring devices, such as photodiodes, photomultipliers or CCD cameras. That method is disclosed, for example, in WO 95/33197. It is also possible for the portion of evanescently excited radiation coupled back into the waveguide to be coupled out by means of a diffractive optical element, for example a grating, and measured. That method is described, for example, in WO 95/33198.
In affinity sensory analysis, for specific identification of an analyte in a sample, which may consist of a complex mixture of substances, and for binding the analyte molecules to the surface of the waveguide, in the region of the penetration depth of the evanescent field, biochemical recognition elements are immobilized on the waveguide surface either directly or by means of an adhesion-imparting layer. For the purpose of detecting the analyte, the dissolved sample is brought into contact, either in intermittent flow or continuous flow, with the recognition elements immobilized on the waveguide surface.
A problem that arises when using measuring cells in which the sample liquid comes into contact with the diffractive coupling-in elements is that the conditions for coupling-in of the excitation light may change as a result of molecular adsorption or binding onto the coupling-in elements. Furthermore, since unbound luminescent or fluorescent molecules are excited by the portion of excitation light that is not coupled into the waveguide but enters the solution unrefracted at zero order, background luminescence or fluorescence may be excited deep within the sample, some of which may be coupled into the waveguide via the coupling-in grating and may impair the accuracy and sensitivity of the determination of the analyte.
Isolating the coupling-in element from the region of contact with the sample by means of materials on which no special demands are placed in terms of transparency and refractive index may result in significant impairment in guidance of the light, possibly even in its entire suppression, in the region of contact with the sample, which is the region relevant to the measurement. That problem is described in greater detail in WO 97/01087.
In order to reduce those disadvantages, WO 97/01087 describes a counter-flow cell in which a transparent reference liquid, which is guided in countercurrent to the analyte sample and does not interact specifically with the recognition elements, is used to produce a sample-free blocking volume in the region of coupling-in elements of optical waveguides, so that constant conditions are obtained in the coupling-in region of the excitation radiation. However, that arrangement, improved especially for the measurement of evanescently excited radiation, is technically relatively complex, is scarcely feasible for intermittent flow applications and accordingly is poorly suited, from the point of view of ease of operation, to routinely use in, for example, diagnostic devices.
Analytical Chemistry, Volume 62, No. 18 (1990), pages 2012-2017, describes a flow-through flow cell made from silicone rubber and applied to an optical waveguide having a coupling-in grating and a coupling-out grating. The coupling-in element and coupling-out element are located in the region of the sample flow channel. Using that arrangement, changes in light absorption and refractive index are measured without selective interaction with specific recognition elements at the waveguide surface. In the case of the analyte (a dye solution in the case of absorption-dependent measurement, or liquids having different refractive indices in the case of refractive index-dependent measurement) adsorption phenomena on the surface are disregarded. In those very insensitive measurements, the changes in the effective refractive index that are to be expected for a mode guided in the waveguide are in fact negligible compared with the large changes in the refractive indices of the solutions fed in--even in the event of a monolayer of molecules being adsorbed--which is contrary to the disruptions to be expected in the very much more sensitive method of determining the luminescence generated in the evanescent field. Of course, in the case of the refractive index-dependent measuring method based on the change in the coupling-in or coupling-out angle, contact between the sample and the coupling elements is actually necessary in order to generate the measurement signal. As a result of that configuration, which has a coupling-in element and coupling-out element located within the sample flow channel, the sample cell merely has the task of providing a seal against the efflux of liquid without any further demands being placed on optical properties of the material.
In order to carry out analyte determinations that are based on measuring the luminescence generated in the evanescent field of a waveguide, a device is therefore required by means of which radiation evanescently excited through a planar optical waveguide can be determined with a high degree of consistency and measurement accuracy, the measuring device at the same time being easy to produce and easy to operate.
It has now been found, surprisingly, that
a) a high degree of measurement accuracy is achieved, PA1 b) an excellent degree of measurement consistency is achieved, PA1 c) a high degree of measurement sensitivity is achieved, PA1 d) it is possible for the device filled with analyte molecules, especially immobilised analyte molecules, to be stored for a relatively long time, and PA1 e) devices that are easy to operate in terms of measurement technique are made available, when the device comprising a waveguide and sample receptacle is so configured that the diffractive element for coupling in excitation radiation is fully covered, at least in the coupling-in region of the excitation radiation, by a layer that is transparent to the excitation radiation and to the evanescently excited radiation.
In view of the fact that the recess is arranged downstream of the coupling-in element in the direction of propagation of excitation radiation and that the diffractive element (coupling-in element) is covered by a layer forming the recess, constant coupling-in conditions are obtained for the excitation radiation in the coupling-in element. On the other hand, abrupt changes in refractive index in the region of penetration of excitation radiation into the material adjacent to the waveguide are minimized to a very large extent. Substantially disruption-free guidance of the excitation radiation and evanescently excited and coupled-back radiation in the waveguide is achieved, which results in a high signal yield and, for example, even under investigation conditions that are frequently not entirely optimal in routine use yields analytical results that can still be utilized. Disruptive abrupt changes in refractive index are also suppressed by means of the rounding.